Hepatitis caused by infection with HCV (hepatitis C virus) becomes chronic with high incidence, and as the infection period is prolonged it often progresses to liver cirrhosis and hepatocellular carcinoma. However, since infection with HCV occurs mainly through blood and blood-derived components, it is possible to identify and eliminate the source of infection to block the infection route. Current methods of identifying infection sources are primarily methods of detecting antibodies against HCV polypeptides, but methods are being sought that can identify infection sources with greater accuracy.
Such methods are being sought because of the existence of a period of time known as the “window period” after HCV infection during which the antigen is present but antibodies are not yet produced. Antibody testing cannot determined whether serum taken during this period is infected or not. Therefore, there is a risk of secondary infection by the blood derived components, such as blood donation, blood components, factors from blood, contaminated specimens in the window period, because blood donor is screened by the antibody test that can not exclude such specimens. For this reason it has been necessary to detect HCV itself, that is, HCV particles, instead of antibodies against HCV polypeptides to reduce the risk.
Detection and measurement of HCV itself is possible by detecting antigens or genome (RNA) in the HCV particles. Here, an antigen in the HCV particles could be core antigen or an envelope antigen (E1, E2).
A lot of vanants were reported in the antigenic region of envelope protein such as hyper variable region. In addition, heterogeneities of sequences between genotypes were reported. In order to detect all of these variants and heterogeneous sequences, it is necessary to use probes that bind to several regions respectively.
Here, “probe” will be used to refer to a molecule that binds specifically to an antigen, for example a molecule which recognizes and binds to an antigen molecule, such as a receptor, antibody, recombinant antibody, functional molecule or functional structure.
The amino acid sequences of core antigen were more conserved than those of envelope antigens. By selecting well conserved regions among several HCV genotypes, a probe recognizing core antigen of all genotype could be obtained. Consequently, the method whose results should not be affected by genotypes will be constructed.
However, one point must be considered in constructing systems for detection of antigens. Specifically, it is highly possible that antibodies in specimens from the subject compete with the antigen-detecting probe for the binding sites, resulting in lowering the detection of sensitivity for the antigen by interfering probe binding. A method would be constructed by using probes recognizing the regions that could not be bound or interfered by antibodies in the specimens. However, it is difficult to prepare probes that fulfill these conditions for molecules reported to have multiple antibody-binding sites, such as HCV core antigen.
Thus, detection of antigen molecules requires elimination of antibodies that inhibit probe binding. Methods of elimination include methods of elimination based on physical principles, for example methods utilizing differences in molecular weight for separation and fractionation of HCV particles and antibodies. Examples of such methods include gel filtration, ultracentrifugation, density gradient centrifugation and molecular weight fractionation using membranes such as ultrafiltration membranes. However, since antibodies often form complexes with other biomolecules whereby they become high molecular weight entities, their separation from HCV particles is difficult by methods based on physical principles. These methods also employ special equipment during the processing steps, which makes their application difficult for mass screening, such as blood screening.
HCV particles are preferentially precipitated by the difference of their solubility in water-based solution containing PEG (polyethylene glycol), which alter the microenvironment of water. However, it will be very difficult to separate antibodies and their complex with antigens from HCV particles, because these components precipitated in same fractions. Moreover, HCV particles often form immune complexes between the antigens in the HCV particles and antibodies that recognize them, and it is difficult to separate only the antibodies or antigens from the immune complexes.
The methods implemented are therefore ones whereby substances (antibodies, etc.) that inhibit probe functions are eliminated by destroying their functions. One such method for losing antibody functions is a method in which the antibody protein is denatured by exposure to conditions that denature the protein structure, but it is essential here to destroy the function of the antibody while not eliminating the function of the object antigen, i.e. the function of binding with the probe, which means not losing the epitope or allowing the epitope to be displayed again, if the probe is an antibody.
The target function of a method of determining the HCV infection will differ depending on the goal.
Antibody testing is a method only to determine the specimen containing antibodies against HCV. When antibodies against HCV are present in a specimen, there are cases where the specimen donor is currently contains HCV because of active injection of HCV, while there are other cases where the specimen does not contain HCV because of HCV elimination from body by treatment or material recovery it is difficult to discriminate these therefore cases based on the presence or absence of antibodies.
The important function of antigen test is to determine whether or not HCV is present in a specimen or to indicate the level of HCV when it is present. It is not dependent on the question of whether or not antibodies are present.
For treatment, HCV antibody testing provides important information for determining whether HCV is the main cause of hepatitis. However, the test for HCV itself is require for definite diagnosis. Determining whether HCV has been eliminated from the body is important in judging the efficacy of treatment. The information of the level of antigen is essential in making a such decision for treatment. That is, for treatment it is important to know whether or not and at what level the antigen is present, regardless of the presence or absence of antibodies. For treatment, then, the most important testing methods are those which indicate the presence or absence and the level of antigen.
For blood and blood-derived components, preventing secondary infection is of greatest importance. For this purpose, testing methods that assess the risk of HCV infection. Antibody testing is currently being used as the primary testing method in this field.
However, as explained above, antibody testing cannot determine whether serum is infected during the window period after HCV infection. Consequently, when blood-derived substances such as transfusion and blood components, blood preparations, etc. are utilized for screening by antibody testing, there is a risk of secondary infection by specimens in the window period.
Coupling this with antigen testing is desired in order to reduce this risk, but antigen testing is still not implemented for mass screening for blood donation.
If a testing method existed that could determine the presence or absence of antigen with a theoretical accuracy (sensitivity, specificity) of 100%, that method could be used as the sole testing method. However any method have a limit of the detection sensitivity and cannot measure levels below that detection sensitivity. Thus, no testing method exists that can discriminate with 100% accuracy. There remains the possibility of missing the source of infection by antigen testing alone, and it is for this reason that measurement of both antibody and antigen is necessary to reduce the risk of secondary infection. If the antigen test, having high sensitivity and specificity sufficient for mass screening, is available, both antigen and antibody test were required for the screening. This requirement will result in higher cost of the screening, because the number of tests performed with samples should increase than present.
It is therefore clear that if measurement of antigen and antibody by the same method becomes possible, it will allow a reduction in the number of tests performed in the field, thus providing a major effect.
As already mentioned, despite the development of antibody detecting methods and antigen detecting methods, when it is attempted to detect antigen under conditions for detecting antibodies as alluded to above, the antigen cannot be efficiently detected because of the presence of antibodies which inhibit binding of probes that detect the antigen. Even under conditions for detection of antigen, however, the adopted methods eliminate antibodies that compete against detection of antigen, as explained above, and therefore antibodies cannot be detected. The currently reported methods, therefore, do not allow detection of antigen and antibody by a single method.